The draft USP chapters <1229> and <1229A> provide guidance on steam sterilization processes. <1229A> focuses specifically on steam sterilization of aqueous liquids, separating it from steam sterilization of parts due to differences in how overprocessing may impact product quality attributes. For liquid sterilization, a dual set of time-temperature parameters is established to assure both sterility and stability. The probability of a non-sterile unit calculation is shown to relate sterilization parameters to achieving the target 10-6 sterility assurance level.

1. In-Process Changes to USP
<1211> Sterilization & Sterility
Assurance of Compendial
Articles
James Agalloco
Agalloco & Associates

2. Disclaimer
This presentation draws on in-process
drafts currently in preparation within USP’s
Microbiology Expert Committee.
The interpretations and emphasis placed
on subjects within this presentation are the
author’s personal opinion and not official
USP positions.
The draft chapters that are issued by USP
in Pharmacopeial Forum on these subjects
differ somewhat from this presentation.

3. Who’s on the Micro Expert Committee?
James Akers, Ph.D., AK&A, Chairman
James Agalloco, A&A
Dilip Ashtekar, Ph.D., Gilead Sciences
Anthony Cundell, Ph.D., Merck & Co.
Dennis Guilfoyle, Ph.D., FDA ORA liaison
Rajesh Gupta, Ph.D., FDA CBER liaison
David Hussong, Ph.D., FDA CDER liaison
Karen McCullough, RMS
Russell Madsen, TWG
Randa Melhem, Ph.D. CBER liaison
Jianghong Meng, Ph.D., University of Maryland,
Leonard Mestrandrea, Ph.D., Mestrandrea LLC
Rainer Newman, Johnson & Johnson (retired)
Mickey Parish, Ph.D., FDA CFSAN liaison
Donald Singer, GlaxoSmithKline
Scott Sutton, Ph.D., Microbiology Network
Edward Tidswell, Ph.D., Baxter
Radha Tirumalai, Ph.D, USP Staff liaison

4. What’s Wrong?
Despite the maturity of the subject, the
practice of sterilization within the global
healthcare industry has descended into rote
repetition of wrong headed expectations.
Regulatory obfuscation and industry apathy
have caused all manner of unnecessary
complications and added patient risks.
Rather than making products safer, we may
have actually increased patient risk!

5. Why did it go Wrong?
We’ve largely ignored the core science that
underlies all sterilization processes.
We have relied on rote repetition of
activities using overly simplistic models and
ignored the core scientific principles upon
which sterilization process must be
constructed.
If we haven’t made it sterilization any
easier, we’ve sure made it dumber.

6. What are the Problems? - 1
Failures to adequately sterilize.
Aseptic processing where terminal
sterilization could be used.
Shortened expiration dates for products.
Increased impurities, particles, extractables.
Wasted energy costs and lost capacity due
to over-processing.

7. What are the Problems? - 2
Ignorance of the objectives.
Oversimplification of key concepts.
Inadequate training of industry.
Confusion among disciplines.
Excess caution built into protocols.
Regulatory excess.
Industry is risk averse to an extreme.
Industry would rather switch than fight!

8. Sterilization Microbiology
101:
What you absolutely must
know in only 5 slides!

9. What’s the Primary Objective?
A minimum PNSU of 10-6 is required.
That means that in routine operation of
the sterilizer, the possibility for a
surviving bioburden microorganism
must be less than 1 in 1,000,000.
It has little to do with the biological
indicator, and even less to do with the
BI population.

10. 106
Microbial Death
103
Curves
100 Biological Indicator
Death Curve
10-3
Here
10-6
Not Here
10-9
n ot a u po P
10-12
i l
10-15 Bioburden
Death Curve
10-18
3 6 9 12 15 18 21 24 27 30
Time

11. 106 D - Value
105
Biological
10
1 log
4
Indicator
103
Death Curve
102
101
not a upo P
100 D value
i l
10-1
10-2
3 6 9 12 15 18 21 24 27 30
Time

12. The D-Value
The D-value is the time required to reduce a
population of microorganisms by one log or a
90% reduction in count.
A D-value is only meaningful if referenced to
specified lethal conditions.
For example D-values should always be
referenced to a temperature, without that
reference they have no meaning, i.e., moist heat
D121.1°C or dry heat D170°C.
For D-values in gases / liquids the agent
concentration, RH and temperature must be
indicated, i.e., D900 PPM, 75% RH,30°C

13. Calculation of PNSU (SAL)
−F
log N u = + log N 0
D
where:
Nu = SAL / PNSU
D = D-value of the natural bioburden
F = F-value (lethality) of the process
N0 = bioburden population

14. What’s Been Missing?

15. Impact of Sterilization
A balance must be achieved between the
need to maintain a safe, stable and
efficacious product while providing sufficient
lethality to attain a minimum level of sterility
assurance.

16. The Forgotten Objective
Achieving sterility (aka minimum PNSU) is
only half of what must be accomplished.
In order to use the materials after the
process their essential quality attributes
have to be maintained.
We can most definitely have too much of a
good thing. If in the effort to kill
microorganisms, we do lasting physical or
chemical damage to the items being
sterilized we have accomplished nothing of
value.

17. Consequences of Over-
processing
Reduce potency
Increased degradation
Increase in extractables / leachables
Increase in particles – visible & sub-visible
Loss / weakening of package integrity
Appearance changes
Changes in physical properties
Limited growth promotion (lab media)
And most important of all – using an
aseptic process instead of a terminal
sterilization process.

18. What is USP’s Role?

19. USP’s General Chapters
USP intends that General Chapters above 1000
will be entirely informational and will not contain
monograph-related requirements. Topics that might
be covered in informational chapters include:
 Background, theory, and future directions/applications
 Areas that need standards, e.g., nanotechnology
 Safety approaches and information
 Guidance chapters for good food and drug practices
 Drug development and registration documents
 Supply chain management documents, including GMP
analyses and comparisons
 Comparisons across the USP compendia.
General Chapter Management in the 2010–2015 Cycle, PF, Vol. 35, 5, Sept-Oct 2009

20. USP <1211> & Related Content
The core of this chapter dates to the 1980’s
and the supportive elements were developed
to address specific needs.
The combined set of chapters is a patchwork
quilt of somewhat disconnected ideas.
Additionally, technological advances have
made some of the content out of date.
We did a quick fix in 2010 to fix the biggest
problems and outlined a plan for a complete
revision of all sterilization & sterility
assurance related content.

21. <1211> The Planned Revision
Started Here: Sterilization at a more basic level: more
instruction, less standardization
 Individual chapters on each sterilization method: allows for easier
revision.
 Separate gas & vapor sterilization; Separate dry heat sterilization &
depyrogenation; separate steam for parts and liquid filled
containers; none of these are really the same process
 New chapters on chemical sterilization: no prior information
 Aseptic processing as a separate chapter: not strictly a sterilization
subject; needs better connection to other supportive chapters
 Update references throughout. New definitions for sterilization
validation models. Clarify the role of the biological indicator. Clarify
PNSU, SAL and risk to patient.
 Integrate Endotoxin Indicator chapter as well as BI & CI content.
 Move BI monographs out of “official chapters”.
 Allow for easier development of other needed content in future.
 Depyrogenation treated independently of sterilization
Finished Here: Separation of Sterilization, Depyrogenation
and Sterility Assurance content.

22. The Game Plan
Revise the entire content, separating it for
ease of development, review, approval and
roll out.
The current content will remain in place and
be revised in piece meal fashion.
Sterilization process specific content has
been given priority, but supportive content
revision is also underway.
The first draft of chapters will appear in
Pharmacopeial Forum beginning in 2012.

23. Old & New Structure Overview
<1211>Sterilization & Sterility Assurance of
Compendial Articles will be divided into three major
areas:
 <1211> General Concepts for Sterility Assurance
 Aseptic Processing, Environmental Monitoring, Sterility Testing,
Parametric Release, & other general sterility assurance related
content
 <1229> General Concepts for Sterilization
 Sterilization processes, BI, CI’s, other sterilization related content
 <1228> General Concepts for Depyrogenation
 Depyrogenation processes, EI’s, other related content
Work on <1211> section has been deferred
because it is believed that sterilization &
depyrogenation chapter revisions are more
urgently needed.

24. What’s been completed so far

25. Where are we now?
The planned structure is largely defined.
We are working on multiple tracks
 Sterilization Process chapters – with most used
first
 Steam for Parts / Hard Goods
 Steam for Liquid Filled Containers
 Sterilizing Filtration
 Radiation Sterilization
 Gas Sterilization
 Sterilization Support chapters beginning with
 Bioburden Monitoring
 Biological Indicators (starting soon)
 Depyrogenation processes – starting soon &
independent of the sterilization effort

26. <1229> Introductory Chapter
Provides an overview and introduces
common elements related to all sterilization
methods. Includes:
 Establishing & Justifying Sterilization
Processes
 D-value and Microbial Resistance
 Biological & Physical Data
 Sterilization Indicators & Integrators
 Selection of an Appropriate Method
 Routine Process Management

27. <1229> The Main Point
“It is generally accepted that sterilized
articles or devices purporting to be
sterile attain a 10–6 microbial survivor
probability, i.e., assurance of less than
1 chance in 1 million that viable
bioburden microorganisms are
present in the sterilized article or
dosage form.”

28. Overkill Sterilization
106
103 Complete
destruction of the
100
Biological Indicator
10-3 at this time point
10-6
10-9
P
n
u
p
o
a
t
i
l
Results in Overkill of the
10-12
bioburden to the PNSU
10-15 where these line intersect
10-18
3 6 9 12 15 18 21 24 27 30
Time

29. BB/BI with 10 BI Challenge 6
106
103 Partial destruction of the
BI at this point
100
10-3
10-6 Destruction of the Bioburden
where these lines intersect
10-9
P
n
u
p
o
a
t
i
l
10-12 PNSU
10-15
10-18
3 6 9 12 15 18 21 24 27 30
Time

30. BB/BI with <10 BI Challenge 6
106
103 Complete destruction of
the BI at this point
100
10-3
10-6 Destruction of the Bioburden
where these lines intersect
10-9
P
n
u
p
o
a
t
i
l
10-12
PNSU
10-15
10-18
3 6 9 12 15 18 21 24 27 30
Time

31. Bioburden Method
106
103 Complete destruction of
the bioburden challenge
100
at this point
10-3
10-6
10-9 Destruction of the lot
P
n
u
p
o
a
t
i
l
PNSU
bioburden
10-12 where these lines intersect
10-15
10-18
3 6 9 12 15 18 21 24 27 30
Time

32. Steam Sterilization

33. <1229S> Direct Steam Sterilization
Separated prior sub-chapter into parts
<1229S> and liquids <1229A> to allow for
differences, and greater clarity.
“overkill approach” is the method of choice.
Separates processes where over-processing
is not a concern from those where it is.
In theory parts sterilization has no upper
limit, while terminal / liquid sterilization is
bounded both above and below the desired
process.

34. Parts vs. Liquid Sterilization
Heat Input 1229A
1229S
Sterile
Non-Stable
Sterile
Sterile
Stable
Non-Sterile Non-Sterile
t ae Ht c ei D
Stable
r
t c ei dn
r I
Product Quality Attributes Product Quality Attributes
Non-issue - Overkill Method An issue – BB/BI Method

35. <1229A> Steam Sterilization of
Aqueous Liquids
The method of choice for liquid parenteral
products, and similar processes are utilized for
laboratory media and process intermediates.
“Where the overkill approach can be utilized for
terminal sterilization of sealed liquid containers, it
is the preferred approach.”
“a dual set of requirements is established for
nearly every important processing parameter.
Sterilization time-temperature or F0 conditions will
include both lower (sterility related) and upper
(stability related) limits to simultaneously assure
safety and efficacy of the processed materials.”

36. <1229A> Steam Sterilization of
Aqueous Liquids
Terminal sterilization of products
 High/Low F0, variety of BI’s, Overkill & BB/BI
method
Media for laboratory usage
 High/Low F0, Overkill & BB/BI method, BI
usage? - self indicating?
Intermediates / process aides
High/Low F0, BI usage, Overkill & BB/BI method
Laboratory and production bio-waste
Low F0, G. stearothermophilus, Overkill method,
condensate collection / kill

37. <1229A> Steam Sterilization of
Aqueous Liquids
Probability of a Non-Sterile Unit (PNSU)
−F
log N u = + log N 0
Where D
Nu = Probability of a Non-Sterile Unit
D = D-value of the natural bioburden
F = F-value of the process
N0 = bioburden population per container
Validation Routine Usage
F0 = 8.0 minutes F0 = 8.0 minutes
D121 of BI = 0.5 minutes D121 of bioburden = 0.005 minutes
N0 of BI = 106 N0 of bioburden = 100 ( or 102)
PNSU for BI = 10-10 PNSU for Bioburden = 10-1,598

38. Sterilization by Filtration

39. Sterilization by Filtration Draft
Sterilizing filtration is a retentive process,
not a destructive one.
Physical removal of microorganisms
depends on the upstream bioburden, the
properties of the solution, the filtration
conditions and the filter itself.
Can be validated to consistently yield
solutions that are sterile as defined in
<1229>.

40. Sterilization by Filtration - 1
Definition and description of “sterilizing-
grade filter”
Retention mechanisms and factors
affecting retention
 Nature of “pores” and microorganisms
 Composition and structure of filter matrix
 Composition of filtered solution
 Filtration conditions
Filter efficacy
 Log-reduction value

41. Sterilization by Filtration - 2
Validation
Integrity test principles and methods
 Bubble point
 Diffusive flow
 Pressure hold
Pre- and post-filtration and sterilization
integrity testing
The need for pre-filtration bioburden control
Troubleshooting common filtration
problems

42. Radiation Sterilization

43. <1229R> Radiation Sterilization
“The prevalent radiation usage is either gamma
rays or electron beams. Other methods utilize x-
rays, microwaves and visible light. The impact of
radiation on materials can be substantial and is a
major consideration in the selection of radiation
as a processing method.”
“Radiation sterilization is unique in that the basis
of control … is the absorbed radiation dose, which
can be precisely measured. Dose setting and
dose substantiation procedures are used to
validate the radiation dose required to achieve
sterility assurance level.”

44. <1229R> Radiation Sterilization
The use of BI’s in radiation sterilization is
not necessary:
 Non-spore-formers have been identified as
more resistant than B. pumilus.
 Dose measurement is accurate and has been
closely correlated to microbial destruction.
The dose setting methods of AAMI/ISO are
well established and easily adapted to
pharmaceutical applications. VDmax has been
utilized for terminal sterilization of several
pharmaceutical preparations.

45. VDMAX – Example 1

46. VDMAX – Example 2

47. Gas, Liquid & Vapor Sterilization
Similar but not the same

48. Gas, Liquid & Vapors - D-Values
A D-value is only meaningful if referenced to
specified lethal conditions. For example wet or
dry heat D-values should always be referenced
to a temperature, without that reference they
have no meaning, i.e., D121.1°C or D170°C.
For D-values in gases / liquids the agent
concentration, RH and temperature must be
indicated, i.e., D900 PPM, 75% RH, 30°C
D-values cannot be accurately determined for
vapors.

49. <1229G> Gas Sterilization
Applicable to single phase gaseous
processes only.
 Condensation of the agent is not a consideration
in the execution of these processes.
 Ethylene oxide – model for all systems
 Chlorine dioxide
 Ozone
Two validation approaches defined
 Traditional half-cycle method
 Bracketing method – variations in concentration,
relative humidity and temperature. More efficient
& more scientific as well.

50. 106
Half Cycle Approach
103 Death Curves
100 Half Cycle
Kill of
10-3 Bioindicator
10-6
Half
Cycle
10-9
not a upo P
10-12
i l
10-15
Full Cycle Half Cycle
10-18 Kill of
Bioburden
3 6 9 12 15 18 21 24 27 30
Time

51. 106
103 Bracketing Approach
100
Death Curves
“w
10-3 or
st
ca
se
X Validation Cycles
”m
10-6 at
eria
10-9 X
l
sc
not a upo P
yc
“w
le
or
ro
st
ut
10-12 ca
in
se
e
st
”
i l
st
er
10-15 er
ili
Routine il
za
iz
X at
tio
io
Process n
n
10-18 cy
cy
cl
cl
e
e
3 6 9 12 15 18 21 24 27 30
Time

52. <1229L> Liquid Sterilization
Chemical Sterilants in aqueous solutions
 Aldehydes – CH2O, CH3CHO, etc.
 Acids – HNO3, H2SO4, peracetic, etc.
 Bases – NaOH, KOH, etc.
 Oxygenating compounds – H2O2, O3, ClO2, etc.
 Halides – NaOCl, Cl2, etc.
Must include an aseptic post-cycle quench step to
stop process prior to adverse material impact.
Validation like gas sterilization. The phase is
different but the same parameters apply.

53. Gas/Liquid vs. Vapor Sterilization
Gases are more penetrating, liquids more uniform in
concentration, and both are less subject to variations in
temperature and relative humidity.
Vapors will have different concentrations in each phase.
When a vapor has 2 possible condensable components it
is even more difficult to predict conditions anywhere.

54. <1229V> Vapor Sterilization
Intended for condensing vapor systems (gas and
liquid phases present simultaneously)
 Hydrogen Peroxide
 Peracetic Acid
The presence of multiple phases simultaneously
complicates concentration determination at the
point of sterilization.
D-value determination is problematic because of
difficulties with parameter measurement in a multi-
component 2 phase system.
Approaches for validation are a hybrid of the liquid
and gas sterilization methods.

55. <1229V> Vapor Sterilization
The kill rates in the gas and liquid phase are
different reflecting the different concentrations and
moisture present in each phase.
The conditions within a vapor system are unlikely
to be constant & uniform because the agent supply
is at a higher temperature than the chamber.
The conditions at any location may change during
the course of the process.
Reproducible kill is possible despite all of the
complication because the agent is lethal in both
phases;
It’s more complex than any of the other methods.

56. <1229V> Vapor Sterilization
Two validation approaches can be utilized, with
the only supportive evidence derived from
microbial destruction.
 Traditional half-cycle method
 Bracketing method
The linearity of microbial destruction cannot be
assured as the process conditions may not be
completely homogeneous.
The efficacy of the agents used should assure
sterilization, however we do not have the ability to
predict the outcome because the process
parameters may vary substantially across the
chamber.

57. 106
Bi-Phasic Kill Possibilities
103
100 Gas Phase
Throughout
10-3 Gas Phase Early
Liquid Phase Late
10-6
The difference
10-9 Liquid Phase Early
not a upo P
in kill rates Is
Gas Phase Late
10-12 unknown
i l
10-15
10-18 Liquid Phase
Throughout
3 6 9 12 15 18 21 24 27 30
Time

58. 106
103 Bi-Phasic Death Curves
100
10-3 Gas Phase Death Curve
10-6
10-9
not a upo P
“w
or
st Liquid Phase Death Curve
10-12
ca
Composite Death Curve
se
i l
”
10-15
ma
te r
ial
10-18
s cy
cle
3 6 9 12 15 18 21 24 27 30
Time

59. 106
103
Vapor Process
Bracketing Approach
100 Death Curves
“w
ors
10-3 tcas X Validation Cycles
e”
10-6
ma
ter
X
i
a ls
10-9
not a upo P
cyc
“w “w
or or
ro r
le
st st
uou
10 ca
tint
-12
se
ca
ene
i
”
se
st s
i l
st
erte
er
”
10-15 ili
iliri
ma
Routine z
zaiz
X at
l
te r
io
tiat
ono
Process n
ial
cy
i
10-18
cyc
n
cl
sc
e
clyc
yc
e le
le
3 6 9 12 15 18 21 24 27 30
Time

60. Dry Heat Sterilization

61. <1229H> Dry Heat Sterilization
Distinction has been made between dry heat
sterilization and depyrogenation because of major
process differences.
Dry heat sterilization:
 Is almost always performed in ovens in a batch
process.
 Uses a biological indicator B. atrophaeus.
 Is usually in the 160-180°C temperature range.
 A reasonable mathematical correlation between
physical data and microbial effect exists.
Physical requirements are less definitive than for
steam processes, but still apply.
Dry heat depyrogenation will be in a separate
chapter.

62. <1229> Bioburden Monitoring
Reviews the relevant concerns for
bioburden content
 Ability to survive the process
 Population
 Risk to Public Health
Considers patient & product impact
Provides a decision tree for use in
establishment of a monitoring program.

64. Depyrogenation

65. <1228> Depyrogenation Methods
<1228D> – Dry Heat Depyrogenation
<1228C> – Chemical Depyrogenation
<1228F> – Depyrogenation by Filtration
<1228P> – Depyrogenation by Physical
Means
All of these need integration with endotoxin
indicator and testing chapters.

66. <1228D> Dry Heat Depyrogenation
Differs from dry heat sterilization in several ways:
Dry heat depyrogenation:
 Predominantly utilized for glass and stainless steel
items.
 Batch and continuous processes are in use.
 An endotoxin monograph has been completed and will
be inserted within the overall <1228> revision.
 Usually in the >200-300°C temperature range.
 Mathematical correlation between physical data and
microbial effect is extremely poor. Defined physical
parameters have proven problematic.
Endotoxin destruction is the primary goal.

67. <1229?> Biological Indicators
Major changes are anticipated for USP’s
existing biological indicator content.
Bring together in one comprehensive
chapter the content currently found in:
 <1211> Sterilization & Sterility Assurance of
Compendial Items
 <1035> Biological Indicators for Sterilization
 <55> Biological Indicators —Resistance
Performance Tests
And

68. <1229?> BI Monographs
 Biological Indicator for Dry-Heat Sterilization,
Paper Carrier
 Biological Indicator for Ethylene Oxide
Sterilization, Paper Carrier
 Biological Indicator for Steam Sterilization, Paper
Carrier
 Biological Indicator for Steam Sterilization, Self-
Contained
 Biological Indicators for Moist Heat, Dry Heat, and
Gaseous Modes of Sterilization, Liquid Spore
Suspensions
 Biological Indicators for Moist Heat, Dry Heat, and
Gaseous Modes of Sterilization, Nonpaper

69. The Rest of the Story

70. Old & New Structure Overview
<1211> Existing Sterilization & Sterility
Assurance of Compendial Articles will be
divided into three major areas:
 <1211> General Concepts for Sterility
Assurance
 Aseptic Processing, Environmental Monitoring,
Sterility Testing, Parametric Release, & other general
sterility assurance related content
 <1229> General Concepts for Sterilization
 Sterilization processes, BI, CI’s, other sterilization
related content
 <1228> General Concepts for Depyrogenation
 Depyrogenation processes, EI’s, other related content

71. <1211> Introductory Chapter
General Concepts for Sterility Assurance
Address the relationship between sterility, and
sterilization, tying in sterilization, aseptic
processing, environmental monitoring and sterility
testing to provide greater clarity.
Sterilization process controls -> sterility
(terminally sterilized products)
Sterilization process controls (multiple processes)
-> aseptic processing / environmental monitoring
-> sterility (aseptically produced products)

72. Planned New Chapters in 1211?
Revised content on aseptic processing
(currently in 1211, but minimally detailed).
Needs linkage to revised <1116>
Microbiological Control and Monitoring of
Aseptic Processing Environments.
New content necessary on the use of
RABS & isolators for aseptic processing.

73. <1222> Parametric Release of
TS
Aligns the guidance with global regulatory
expectations.
Must be aligned with <1229>,<1229T>,
<1229G>,<1211H> and <1229R> as these
chapters evolve because all of these
sterilization chapters are relevant for
parametric release.

74. Planned New Chapters in 1211?
1211T – Terminal sterilization perspectives
covering all process types (combination
with 1222 is possible).
1211P – Post Aseptic Fill adjunct treatment
using either radiation or moist heat.
Integrate <1207> Container-closure
integrity with possible new content in
<1211> and <1229>. Microbiology Expert
Committee members are involved with the
<1207> revision.

75. Many THANKS for YOUR Attention
Dziękuję Ďakujem dhanya-waad Дякую
bedankt ‫תודה‬ go raibh maith agat
tesekkürle Спасибо ‫ شكرا‬Thank yu
ً‫ ا‬tack så mycket
Merci köszi díky
Thank you faleminderit
Shukriyâ Danke hvala kiitos
takk Obrigada Mulţumesc nandri
Grazie anugurihiitosumi
Ευχαριστώ dhanya-waad köszönöm
tack
Muchas gracias ačiû Terima Kasih
aitäh děkuji vam mange tak salamat